Biomedical subjects

Ravshan Z Sabirov

Publications and source records attributed to Ravshan Z Sabirov.

12 recordsLinked to original sources

PubMed2006

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress.

Astrocytes release glutamate upon hyperexcitation in the normal brain, and in response to pathologic insults such as ischemia and trauma. In our experiments, both hypotonic and ischemic stimuli caused the release of glutamate from cultured mouse astrocytes, which occurred with little or no contribution of gap junction hemichannels, vesicle-mediated exocytosis, or reversed operation of the Na-dependent glutamate transporter. Cell swelling and chemical ischemia activated, in cell-attached membrane patches, anionic channels with large unitary conductance (approximately 400 pS) and inactivation kinetics at potentials more positive than +20 mV or more negative than -20 mV. These properties are different from those of volume-sensitive outwardly rectifying (VSOR) Cl- channels, which were also expressed in these cells and exhibited intermediate unitary conductance (approximately 80 pS) and inactivation kinetics at large positive potentials of more than +40 mV. Both maxi-anion channels and VSOR Cl- channels were permeable to glutamate with permeability ratios of glutamate to chloride of 0.21 +/- 0.07 and 0.15 +/- 0.01, respectively. However, the release of glutamate was significantly more sensitive to Gd3+, a blocker of maxi-anion channels, than to phloretin, a blocker of VSOR Cl- channels. We conclude that these two channels jointly represent a major conductive pathway for the release of glutamate from swollen and ischemia-challenged astrocytes, with the contribution of maxi-anion channels being predominant.

Animals↗

PubMed2005

Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins.

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Adenosine Triphosphate↗

PubMed2004

Detecting ATP release by a biosensor method.

Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

Adenosine Triphosphate↗

PubMed2004

Sizing the pore of the volume-sensitive anion channel by differential polymer partitioning.

Partitioning of ethylene glycol and its polymeric forms into the pore of the volume-sensitive outwardly rectifying (VSOR) anion channel was studied to assess the pore size. Polyethylene glycol (PEG) PEG 200-300 (Rh = 0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh = 0.62-1.91 nm) had little or no effect. Since all the molecules tested effectively decreased electric conductivity of the bulk solution, the observed differential effects between PEG 200-300 and PEG 400-4000 on the VSOR single-channel current are due to their limited partitioning into the channel lumen. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm.

Anions↗

PubMed2004

Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions.

It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin-luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (approximately 390 pS). The channel was selective to anions and showed significant permeability to ATP4- (PATP/PCl approximately 0.1) and MgATP2- (PATP/PCl approximately 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels.

Adenosine Triphosphate↗

PubMed2004

Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway.

The newly proposed function of the maxi-anion channel as a conductive pathway for ATP release requires that its pore is sufficiently large to permit passage of a bulky ATP(4-) anion. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the first time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cutoff radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of approximately 1.3 nm. These three estimates are considerably larger than the radius of ATP(4-) (0.57-0.65 nm) and MgATP(2-) (approximately 0.60 nm). We therefore conclude that the nanoscopic maxi-anion channel pore provides sufficient room to accommodate ATP and is well suited to its function as a conductive pathway for ATP release in cell-to-cell communication.

Adenosine Triphosphate↗

PubMed2004

ATP-conducting maxi-anion channel: a new player in stress-sensory transduction.

The regulated release of ATP is a fundamental process in cell-to-cell signaling. The electrogenic translocation of ATP via an anion channel has been suggested as one possible mechanism of the release. In this review, we survey possible candidate channels for this pathway. The maxi-anion channel characterized by an exceedingly large unitary conductance has been a stray channel with regard to its function. A newly discovered property, its ATP conductivity and its activation in response to stress signals, indicates that this channel has a central role in stress-sensory transduction for cell volume regulation and tubuloglomerular feedback.

Adenosine Triphosphate↗

PubMed2003

Calcium-activated nonselective cationic channel in macula densa cells.

Patch-clamp experiments in cell-attached (c/a) and inside-out (i/o) configurations were performed to directly observe ionic channels in lateral membranes of macula densa (MD) cells from rabbit kidney. In the presence of 140 mM KCl in the pipette and normal Ringer solution in the bath, we repeatedly observed in c/a and in i/o configurations a 20- to 23-pS channel with a linear current-voltage (I-V) relationship reversing near 0 mV. Ionic replacement in the bath solution clearly indicated a cationic selectivity but with equal permeability for Na+ and K+. Single-channel kinetics was characterized by higher open probability at positive membrane potentials. In i/o experiments, elimination of bath Ca2+ (<or=1 microM) abolished channel activity in a reversible manner. This MD nonselective cationic channel was found to display a certain Ca2+ permeability because single-channel events could be detected when the pipette potential was very negative (-60, -80, and -100 mV) in the presence of 73 mM CaCl2 in the bath solution. The similarities between this channel and some channels of the transient receptor potential family suggest a possible role for this MD basolateral channel in controlling membrane potential and regulating Ca2+ entry during MD cell signaling.

Animals↗

PubMed2003

Upregulation of swelling-activated Cl- channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells.

Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl- currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl- currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl- currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl- current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl- currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 micro M) was lower than that in control cells (31 micro M). However, the VSOR Cl- currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was markedly diminished by tyrphostin B46 but not significantly affected by tyrphostin AG1296. We thus conclude that the EGF receptor tyrosine kinase upregulates the activity of the VSOR Cl- channel, mainly by enhancing the volume sensitivity.

Algorithms↗

PubMed2003

Ischemia-induced enhancement of CFTR expression on the plasma membrane in neonatal rat ventricular myocytes.

Pathophysiological functions of cardiac cystic fibrosis transmembrane conductance regulator (cCFTR) in ischemia are not well known. Using neonatal rat ventricular cardiomyocytes in primary culture in this study, we thus examined whether the CFTR protein is expressed and is functioning as a cAMP-activated anion channel on the plasma membrane under ischemic conditions. After the cells were subjected to simulated ischemia (O(2) and glucose deprivation), an up-regulation of the CFTR expression was transiently observed in the membrane fraction by Western blot. A peak expression of mature CFTR protein was found at 3 h of ischemia, and thereafter the signal diminished gradually. In contrast, the results of Northern blot indicated that the expression level of CFTR mRNA changed little until 3 h of ischemia, whereas the level slightly decreased after 8 h of ischemia. An immunohistochemical examination showed, in agreement with the results of Western blot analysis, that the expression of CFTR protein on the plasma membrane became most prominent at 3 h of ischemia, whereas the plasmalemmal CFTR signal was markedly reduced after 8 h of ischemia. Whole-cell recordings showed that the cardiomyocytes responded to cAMP with an activation of time- and voltage-independent currents that contained an anion-selective component sensitive to CFTR Cl(-) channel blockers (NPPB and glibenclamide) but not to a stilbene-derivative conventional Cl(-) channel blocker (SITS). This cAMP-activated Cl(-) channel current was found to be enhanced after an application of ischemic stress for 3 to 4 h. These findings indicate that a plasmalemmal expression of CFTR is transiently enhanced under glucose-free hypoxic conditions presumably because of a posttranslational control.

Animals↗

PubMed2002

Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

Adenosine Triphosphate↗

PubMed2002

Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid.

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with K(d) of 4- 6 microM. The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis-unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans-unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (K(d) of 4- 5 microM) and reduced unitary conductance (K(d) of 13-14 microM) without affecting voltage dependence of open probability. ATP(4-)-conducting inward currents measured in the presence of 100 mM ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.

Adenosine Triphosphate↗